U.S. patent number 7,534,770 [Application Number 10/564,464] was granted by the patent office on 2009-05-19 for glycoside having 4-methylergost-7-en-3-ol skeleton and hyperglycemia improving agent.
This patent grant is currently assigned to Morinaga Milk Industry Co., Ltd.. Invention is credited to Hirotoshi Hayasawa, Ryuuichi Higuchi, Masanori Inagaki, Yousuke Itou, Eriko Misawa, Miyuki Tanaka, Noriko Wakimoto, Muneo Yamada.
United States Patent |
7,534,770 |
Higuchi , et al. |
May 19, 2009 |
**Please see images for:
( Certificate of Correction ) ** |
Glycoside having 4-methylergost-7-en-3-ol skeleton and
hyperglycemia improving agent
Abstract
3-O-.beta.-D-Glucopyranosyl-4-methylergost-7-en-3-ol or a
composition containing 0.001% by mass or more of the aforementioned
compound, which is an extract of a plant of the family Liliaceae
containing the compound or a fraction thereof, is used as an active
ingredient of a hyperglycemia improving agent.
Inventors: |
Higuchi; Ryuuichi (Fukuoka,
JP), Inagaki; Masanori (Fukuoka, JP),
Hayasawa; Hirotoshi (Tokyo, JP), Yamada; Muneo
(Zama, JP), Tanaka; Miyuki (Zama, JP),
Misawa; Eriko (Zama, JP), Wakimoto; Noriko (Zama,
JP), Itou; Yousuke (Zama, JP) |
Assignee: |
Morinaga Milk Industry Co.,
Ltd. (Tokyo, JP)
|
Family
ID: |
35063719 |
Appl.
No.: |
10/564,464 |
Filed: |
March 30, 2005 |
PCT
Filed: |
March 30, 2005 |
PCT No.: |
PCT/JP2005/006019 |
371(c)(1),(2),(4) Date: |
January 12, 2006 |
PCT
Pub. No.: |
WO2005/095436 |
PCT
Pub. Date: |
October 13, 2005 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20080044500 A1 |
Feb 21, 2008 |
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Foreign Application Priority Data
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Mar 31, 2004 [JP] |
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2004-103684 |
Apr 6, 2004 [JP] |
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2004-112108 |
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Current U.S.
Class: |
514/26; 536/6;
424/744 |
Current CPC
Class: |
A61P
7/00 (20180101); A61K 36/896 (20130101); C07J
17/005 (20130101); A23L 33/105 (20160801); A61P
3/10 (20180101); A61K 36/886 (20130101) |
Current International
Class: |
A01N
45/00 (20060101); A61K 36/886 (20060101); C07G
3/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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60-214741 |
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Oct 1985 |
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JP |
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01-312978 |
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Dec 1989 |
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JP |
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05-247086 |
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Sep 1993 |
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JP |
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08-208495 |
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Aug 1996 |
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JP |
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09-040689 |
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Feb 1997 |
|
JP |
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09-059298 |
|
Mar 1997 |
|
JP |
|
09-070278 |
|
Mar 1997 |
|
JP |
|
09-224588 |
|
Sep 1997 |
|
JP |
|
10-036271 |
|
Feb 1998 |
|
JP |
|
10-036283 |
|
Feb 1998 |
|
JP |
|
10-045604 |
|
Feb 1998 |
|
JP |
|
10-120576 |
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May 1998 |
|
JP |
|
10-330266 |
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Dec 1998 |
|
JP |
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11-511482 |
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Oct 1999 |
|
JP |
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2001-520019 |
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Oct 2001 |
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JP |
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2002-205949 |
|
Jul 2002 |
|
JP |
|
2002-371003 |
|
Dec 2002 |
|
JP |
|
2003-048837 |
|
Feb 2003 |
|
JP |
|
2003-095941 |
|
Apr 2003 |
|
JP |
|
2003-286185 |
|
Oct 2003 |
|
JP |
|
WO 97/16438 |
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May 1997 |
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WO |
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WO 99/19505 |
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Apr 1999 |
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WO |
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WO 03/059360 |
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Jul 2003 |
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WO |
|
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English translation. cited by other .
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translation. cited by other .
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Glycemic Control in Diabetes," Diabetes Care, vol. 26, No. 4, pp.
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Extracts," Planta Med., vol. 57, pp. 38-40, 1991. cited by other
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Pulp Extracts on Kidney in Type-II Diabetic Rat Models," Indian
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and B, Glycans of Aloe arborescens var. natalensis Leaves," Int. J.
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Glycemic Control in Diabetes," Diabetes Care, vol. 26, No. 4, pp.
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|
Primary Examiner: Tate; Christopher R
Assistant Examiner: Winston; Randall
Attorney, Agent or Firm: Knobbe, Martens, Olson & Bear,
LLP
Claims
What is claimed is:
1. A purified compound having the following chemical formula (1):
##STR00005##
2. A composition containing 0.001% by dry mass or more of the
purified compound according to claim 1.
3. The composition according to claim 2, which is obtainable from
an extract of a plant of the family Liliaceae or a fraction
thereof.
4. The composition according to claim 3, wherein the plant of the
family Liliaceae is Aloe vera (Aloe barbadensis Miller).
5. A hyperglycemia improving agent, which comprises the compound
according to claim 1 as an active ingredient in a pharmaceutically
acceptable carrier.
6. A drug comprising the hyperglycemia improving agent according to
claim 5.
7. A food or drink comprising the hyperglycemia improving agent
according to claim 5.
8. A food or drink comprising the purified compound according to
claim 1 as an active ingredient, having a hyperglycemia improving
effect, and attached with an indication that the food or drink is
used for improvement of hyperglycemia.
9. A method for the production of a drug for improving
hyperglycemia which comprises mixing the purified compound of claim
1 or a composition containing the purified compound with a
pharmaceutically acceptable carrier ##STR00006##
10. The method according to claim 9, wherein the composition is
obtainable from an extract of a plant of the family Liliaceae or a
fraction thereof containing 0.001% by dry mass or more of the
compound.
11. The method according to claim 10, wherein the plant of the
family Liliaceae is Aloe vera (Aloe barbadensis Miller).
12. A method for improving hyperglycemia, which comprises
administering a compound having a structure represented by the
following chemical formula (1) or a composition containing the
purified compound to a subject whose hyperglycemia is to be
improved ##STR00007##
13. The method according to claim 12, wherein the composition is
obtainable from an extract of a plant of the family Liliaceae or a
fraction thereof containing 0.001% by dry mass or more of the
compound.
14. The method according to claim 13, wherein the plant of the
family Liliaceae is Aloe vera (Aloe barbadensis Miller).
Description
RELATED APPLICATIONS
This application is the U.S. National Phase under 35 U.S.C. .sctn.
371 of International Application PCT/JP2005/006019, filed Mar. 30,
2005, which was published in a language other than English, which
claims priority of JP Application No. 2004-103684, filed Mar. 31,
2004 and JP Application No. 2004-112108, filed Apr. 6, 2004.
1. Technical Field
The present invention relates to a novel glycoside having a
4-methylergost-7-en-3-ol skeleton,
3-O-.beta.-D-glucopyranosyl-4-methylergost-7-en-3-ol, a composition
containing the same, a drug and food or drink containing these.
2. Background Art
4-Methylergost-7-en-3-ol is known to be a substance that exists in
plants (Non-patent document 1). As for prior arts of this compound,
however, there is only a reference concerning the biosynthetic
system of lophenol (one of the stereoisomers of
4-methylcholest-7-en-3-ol) having a structure similar to that of
the aforementioned compound (Non-patent document 2), and the use of
these compounds is not known at all.
The genus Aloe in the family Liliaceae is a group of plants
including Aloe vera (Aloe barbadensis Miller) and Aloe arborescens
(Aloe arborescens Miller var. natalensis Berger) and so forth, and
they are empirically known to have various efficacies. The prior
arts regarding the use of plants of the genus Aloe include
immunomodulating polysaccharides (Patent document 1),
immunosuppression improving agents containing a butanol fraction of
an aloe extract or aloin (Patent document 2), HSP60 family protein
synthesis suppressing agents containing aloin derivatives (Patent
documents 3 to 5), proteins having lectin activity derived from
aloe leaf-skin (Patent document 6) and so forth.
As the prior arts regarding improvement of blood glucose levels by
the plants of the genus Aloe, clinical studies in the United States
(Non-patent document 3) and a hypoglycemic action observed in
animal studies (Non-patent documents 4 and 5) and polysaccharides
in plants of the genus Aloe (Patent document 7) have been
disclosed. In these prior arts, the hypoglycemic ingredients of the
plants of the genus Aloe were predicted to be polysaccharides or
glycoproteins. Furthermore, it has been disclosed that, in a
pressed extract of Aloe vera and a hypoglycemic agent containing
the extract as an active ingredient (Patent document 8), a
characteristic peak unique to an ester group observed in the FT-IR
chart correlates with the activity, that the active ingredient is a
polysaccharide, amino acid, malic acid or the like, and that the
aforementioned active ingredient is degraded in commercially
available Aloe vera gel powders, Aloe vera gel solutions and Aloe
vera gel extracts. Furthermore, in addition to the above, a
hypoglycemic action of aloe polysaccharides (Patent document 9) and
antioxidative action of 7-hydroxychromone contained in aloe (Patent
document 10) have been disclosed.
Meanwhile, because Aloe vera leaf-skin contains barbaloin and
aloe-emodin having a laxative action, it has been conventionally
considered unfavorable for industrial use.
Hemoglobin Alc, a binding product of glucose and hemoglobin,
increases depending on the severity of hyperglycemia in a glucose
level-dependent manner. Because hemoglobin Alc once produced is not
eliminated until the lifetime of erythrocyte (120 days) runs out,
it reflects the past blood glucose control conditions over a long
period of time (Non-patent document 6). Hemoglobin Alc was adopted
as a selected test item of the basic health screening according to
the Health Law for the Aged since 1996 and adopted as an auxiliary
diagnosis indicator of diabetes mellitus in the new diagnosis
criteria of diabetes mellitus in 1999. Therefore, it is considered
that hemoglobin Alc is an indicator of great clinical significance
(Non-patent document 7).
If a hyperglycemic condition is sustained, glucose specific insulin
hyposecretion and insulin resistance are observed and serve as
factors that further aggravate hyperglycemia (Non-patent document
8). Because long-term blood glucose level control is necessary to
prevent progression from the hyperglycemic condition to onset of
diabetes mellitus, it is considered to become necessary to suppress
increase in the hemoglobin Alc level. Alimentotherapies and
exercise are recommended to control blood glucose level in patients
with prediabetes (likelihood of developing diabetes or related
conditions). Although various functional foods for preventing
postprandial increases in blood glucose level (food for specified
health uses) have already been marketed, all of these only have a
temporary effect of suppressing increase in blood glucose level.
Therefore, control of blood glucose level over a long period of
time cannot be expected, and development of such a substance having
a hemoglobin Alc level lowering action as in the present invention
has been desired.
Furthermore, .alpha.-glucosidase inhibitors, sulfonylurea drugs as
insulin secretagogues, thiazolidine derivatives as insulin
resistance improving agents and so forth are currently used as
therapeutic agents for diabetes mellitus. However, the drug
efficacies thereof are not satisfactory, and they suffer many
problems such as side effects causing coma due to rapid drop in
blood glucose level.
Under the aforementioned circumstances, discovery of a substance
that can be safely ingested without causing acute hypoglycemia and
has a long-term blood glucose level control action by decreasing
the hemoglobin Alc level has been strongly desired.
Conventionally, as examples of substances having an effect of
suppressing increases in blood glucose level, the prior art
references have disclosed a hyperglycemia suppressing agent
containing a banaba-derived ingredient (Patent document 11), a
hyperglycemia suppressing agent containing a concentrated extract
of fermentation product of wheats or barleys as an active
ingredient (Patent document 12) and so forth.
Furthermore, as techniques of using a triterpene glycoside as an
active ingredient, for example, an agent for preventing diabetes
mellitus containing a glycoside extracted from Gymnema inodorum as
an active ingredient (Patent document 13), a metabolism improving
method and a composition therefor containing corosolic acid
extracted from banaba as an active ingredient (Patent document 14),
a lipase inhibitor (Patent document 15) and a triterpene derivative
having an immunosuppressing activity (Patent document 16) have been
disclosed.
Furthermore, it has been disclosed that the insulin action
enhancing activity of a compound having a lanostane skeleton or
3,4-secolanostane skeleton (Patent document 17) enhances the
insulin action in regulation of adipocyte differentiation, although
the effect thereof on diseases in the pancreas is unknown.
Furthermore, compounds selected from the group consisting of
24-alkylcholesten-3-ones and 24-alkylcholestan-3-ones that have no
double bond in the basic steroid skeleton have been disclosed as
hypoglycemic agents (Patent document 18).
As for a substance having a similar structure, a glycoside having a
4-methylstigmast-7-en-3-ol skeleton, it has been reported that
3-O-.beta.-D-glucopyranosyl-4-methylstigmast-7-en-3-ol is contained
in Bryony (Bryonia alba), a plant of the family Cucurbitaceae
(Non-patent document 9). However, this is not a plant that has been
generally eaten, and no total synthesis thereof has been reported.
[Patent document 1] International Patent Application Unexamined
Publication in Japanese (Kohyo) No. 2001-520019 [Patent document 2]
Japanese Patent Laid-open (Kokai) No. 08-208495 [Patent document 3]
Japanese Patent Laid-open No. 10-120576 [Patent document 4]
Japanese Patent Laid-open No. 10-045604 [Patent document 5]
Japanese Patent Laid-open No. 10-036271 [Patent document 6]
Japanese Patent Laid-open No. 09-059298 [Patent document 7]
Japanese Patent Laid-open No. 60-214741 [Patent document 8]
Japanese Patent Laid-open No. 2003-286185 [Patent document 9] U.S.
Pat. No. 4,598,069 [Patent document 10] U.S. Patent Application
Publication No. 2003/0207818 [Patent document 11] Japanese Patent
Laid-open No. 2003-095941 [Patent document 12] Japanese Patent
Laid-open No. 2002-371003 [Patent document 13] Japanese Patent
Laid-open No. 05-247086 [Patent document 14] Japanese Patent
Laid-open No. 2002-205949 [Patent document 15] Japanese Patent
Laid-open No. 09-040689 [Patent document 16] International Patent
Application Unexamined Publication in Japanese No. 11-511482
[Patent document 17] Japanese Patent Laid-open No. 10-330266
[Patent document 18] Japanese Patent Laid-open No. 2003-048837
[Non-patent document 1] Chem. Pharm. Bull., pp. 624-626, 1993
[Non-patent document 2] Biochemica Biophysica Acta, pp. 63-88, 2000
[Non-patent document 3] Phytomedicine, Vol. 3, pp. 245-248, 1996
[Non-patent document 4] Phytotherapy Research, Vol. 15, pp.
157-161, 2001 [Non-patent document 5] Phytotherapy Research, Vol.
7, pp. 37-42, 1993 [Non-patent document 6] Nippon Rinsho, No. 748,
Vol. 1, pp. 615-617, 1999 [Non-patent document 7] Nippon Rinsho,
No. 808, Vol. 2, pp. 405-409, 2002 [Non-patent document 8] Yazaki
Y. & Muramatsu M. Ed., "Front Line of Diabetes Mellitus", pp.
126-139, Yodosha 1997 [Non-patent document 9] Khimiya Prirodnykh
Soedinenii, Vol. 3, USSR, 1977
SUMMARY OF THE INVENTION
An object of the present invention is to provide a novel compound
that can be safely ingested without causing acute hypoglycemia and
has a long-term blood glucose level control action to decrease the
hemoglobin Alc level. Another object of the present invention is to
develop a method for producing a composition that does not contain
industrially unfavorable ingredients and contains an effective
amount of the compound from a raw material that can be taken safely
from experiential viewpoint for food and is readily obtained.
The inventors of the present invention assiduously studied in order
to achieve the foregoing objects. As a result, they found that
3-O-.beta.-D-glucopyranosyl-4-methylergost-7-en-3-ol, a novel
glycoside extracted and purified from mesophyll (clear gel portion)
of Aloe vera (Aloe barbadensis Miller), could be safely ingested
without causing acute hypoglycemia and had a long-term blood
glucose level control action for decreasing the hemoglobin Alc
level. The present invention was accomplished on the basis of the
above findings.
That is, the present invention provides a compound having a
structure represented by the following chemical formula (1)
(hereinafter also referred to as "the compound of the present
invention").
##STR00001##
The present invention also provides a composition containing 0.001%
by dry mass or more of the compound of the present invention
(hereinafter also referred to as "the composition of the present
invention"). The composition of the present invention is preferably
an extract of a plant of the family Liliaceae or a fraction
thereof, and according to a particularly preferred embodiment, the
plant of the family Liliaceae is Aloe vera (Aloe barbadensis
Miller).
The present invention further provides a hyperglycemia improving
agent containing the compound or the composition of the present
invention as an active ingredient (hereinafter also referred to as
"the drug of the present invention").
The present invention also provides a drug or food or drink
containing the aforementioned hyperglycemia improving agent.
The present invention also provides food or drink containing the
compound of the present invention or the composition of the present
invention as an active ingredient, having a hyperglycemia improving
effect, and attached with an indication that the food or drink is
used for improvement of hyperglycemia.
Hereinafter, the aforementioned drug and food or drink may be
generically referred to as "the drug, food or drink of the present
invention."
The present invention further provides a method for producing the
compound of the present invention or the composition of the present
invention, which comprises extracting a fraction containing the
compound from a plant of the family Liliaceae containing the
compound, a part thereof or a disruption product thereof by using
an organic solvent or hot water and concentrating the fraction, and
according to a particularly preferred embodiment, the plant of the
family Liliaceae is Aloe vera (Aloe barbadensis Miller).
The present invention further provides use of the compound of the
present invention or a composition containing the same in the
production of a drug for improving hyperglycemia. In the use of the
present invention, the aforementioned composition is preferably an
extract of a plant of the family Liliaceae or a fraction thereof
containing 0.001% by dry mass or more of the aforementioned
compound, and according to a particularly preferred embodiment, the
plant of the family Liliaceae is Aloe vera (Aloe barbadensis
Miller).
The present invention further provides a method for improving
hyperglycemia, which comprises administering the compound of the
present invention or a composition containing the same to a subject
whose hyperglycemia is to be improved. In the method of the present
invention, the aforementioned composition is preferably an extract
of a plant of the family Liliaceae or a fraction thereof containing
0.001% by dry mass or more of the aforementioned compound, and
according to a particularly preferred embodiment, the plant of the
family Liliaceae is Aloe vera (Aloe barbadensis Miller).
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a GC-MS spectrum of the acetylated aglycon moiety of
the glycoside of the present invention (photo to replace drawing: a
half-tone image shown on a display).
FIG. 2 shows a .sup.13C-NMR chart of the acetylated aglycon moiety
of the glycoside of the present invention (photo to replace
drawing: a half-tone image shown on a display).
FIG. 3 shows a graph showing changes over time in random blood
glucose levels of mice administered with the compound of the
present invention.
FIG. 4 shows a graph showing changes over time in fasting blood
glucose levels of mice administered with the compound of the
present invention.
BEST MODE FOR CARRYING OUT THE INVENTION
Hereafter, preferred embodiments of the present invention will be
explained in detail. However, the present invention is not limited
to the following preferred embodiments and can be freely modified
within the scope of the present invention.
The compound of the present invention is a compound having a
structure represented by the aforementioned chemical formula (1),
that is, 3-O-.beta.-D-glucopyranosyl-4-methylergost-7-en-3-ol. That
is, the compound of the present invention has a structure formed by
dehydration condensation of the hydroxyl group at the 3-position of
4-methylergost-7-en-3-ol and the hydroxyl group at the 1-position
of D-glucose.
Furthermore, the composition of the present invention is an extract
from a plant of the family Liliaceae or a fraction thereof
containing 0.001% by dry mass or more, preferably 0.01% by dry mass
or more, more preferably 0.1% by dry mass or more, of the compound
of the present invention. The upper limit of the content of the
compound of the present invention contained in the composition of
the present invention is not particularly limited, and it may be,
for example, 50, 70 or 90% by mass.
The compound of the present invention or a composition containing
the same can be produced by, for example, extracting a fraction
containing the compound of the present invention from a plant
belonging to the family Liliaceae and containing the compound of
the present invention, a part thereof, or a disruption product
thereof by using an organic solvent or hot water and concentrating
the fraction.
Examples of the aforementioned plant belonging to the family
Liliaceae include plants belonging to the genus Aloe or Allium.
Examples of the plants of the genus Aloe include Aloe barbadensis
Miller, Aloe ferox Miller, Aloe africana Miller, Aloe arborescen
Miller var. natalensis Berger, Aloe spicata Baker and so forth. In
the production of the compound of the present invention or a
composition containing the same, although the whole of the
aforementioned plant may be used, it is preferable to use mesophyll
(clear gel portion) thereof. Such a plant or a part thereof is
disrupted preferably by using a homogenizer or the like and thereby
liquefied, and the disruption product is extracted by using an
organic solvent or hot water. Examples of the organic solvent
include alcohols such as methanol, ethanol and butanol; esters such
as methyl acetate, ethyl acetate, propyl acetate and butyl acetate;
ketones such as acetone and methyl isobutyl ketone; ethers such as
diethyl ether and petroleum ether; hydrocarbons such as hexane,
cyclohexane, toluene and benzene; halogenated hydrocarbons such as
carbon tetrachloride, dichloromethane and chloroform; heterocyclic
compounds such as pyridine; glycols such as ethylene glycol;
polyhydric alcohols such as polyethylene glycol; nitrile solvents
such as acetonitrile, mixtures of these solvents and so forth.
Furthermore, these solvents may be anhydrous or hydrous. Among
these solvents, ethyl acetate/butanol mixture (3:1) and
chloroform/methanol mixture (2:1) are particularly preferred.
As the extraction method, a method used for usual extraction of a
plant component can be used. Usually used is, for example, a method
of refluxing 1 to 300 parts by mass of an organic solvent with 1
part by mass of fresh plant or dried plant with heating at a
temperature at or below the boiling point of the solvent and
stirring or shaking, or a method of performing extraction by
ultrasonication at room temperature. By isolating insoluble matters
from the extraction liquor using a suitable method such as
filtration or centrifugation, a crude extract can be obtained.
The crude extract can be purified by various types of
chromatography such as normal or reverse phase silica gel column
chromatography. When a gradient of chloroform/methanol mixture is
used in normal phase silica gel column chromatography as an elution
solvent, the compound of the present invention is eluted with a
mixing ratio of chloroform:methanol=about 5:1. Furthermore, when a
gradient of methanol/water mixture is used in reverse phase silica
gel column chromatography as an elution solvent, the compound of
the present invention is eluted with methanol of a concentration of
about 95%.
The obtained fraction can be further purified by HPLC or the
like.
Whether the compound or composition containing the same obtained as
described above actually contains the compound of the present
invention can be confirmed by, for example, the methods shown in
the examples described later. Whether the compound is a glycoside
bound with glucose at the aglycon moiety, or whether the aglycon
moiety is 4-methylergost-7-en-3-ol can be confirmed by, for
example, .sup.13C-NMR or the like.
The compound of the present invention can also be produced by
condensing D-glucose and 4-methylergost-7-en-3-ol.
4-Methylergost-7-en-3-ol can be obtained by extracting and
purifying it from a plant. D-Glucose and 4-methylergost-7-en-3-ol
can be condensed by, for example, a combination of the methods
described in Jikken Kagaku Koza (Lecture of Experimental
Chemistry), 4th edition, vol. 26, 1992 (described in p. 272, p. 297
and p. 342). That is, D-glucose is completely acetylated, and then
the anomeric position is converted to .alpha.-bromide. Then,
4-methylergost-7-en-3-ol is reacted with .alpha.-bromide in diethyl
ether to attain .beta.-glycosylation, and thereafter the acetyl
group is hydrolyzed in a sodium methoxide/methanol mixture to
obtain the objective compound.
The compound of the present invention has an action of lowering the
hemoglobin Alc level, and as a result, it can control the blood
glucose level over a long period of time. Therefore, it can be used
as an active ingredient of a hyperglycemia improving agent.
Furthermore, because leaf-skin of Aloe vera contains barbaloin and
aloe-emodin having a laxative action, it is conventionally
considered to be unfavorable as a drug, food or drink for which
laxative action is not expected. On the other hand, the composition
of the present invention according to a preferred embodiment can be
obtained by extraction and fractionation from mesophyll (clear gel
portion) of Aloe vera, which can be safely ingested from
experiential viewpoint for food, and therefore it does not contain
barbaloin or aloe-emodin, but contains an effective amount of the
compound of the present invention. Therefore, the composition of
the present invention is also preferred as an active ingredient of
a hyperglycemia improving agent.
The compound or composition of the present invention can be
utilized as an active ingredient of the drug, food or drink of the
present invention as it is. Furthermore, the composition of the
present invention may be a solution and can also be stored and used
as powder after it is lyophilized or spray-dried in a conventional
manner.
As the drug of the present invention, the compound or the
composition of the present invention or those combined with a
pharmaceutically acceptable carrier can be orally or parenterally
administered to a mammal including human. In the drug of the
present invention, the compound of the present invention may be a
pharmaceutically acceptable salt. Examples of the pharmaceutically
acceptable salt include both metal salts (inorganic salts) and
organic salts including, for example, those listed in "Remington's
Pharmaceutical Sciences," 17th edition, p. 1418, 1985. Specific
examples thereof include, but not limited to, inorganic acid salts
such as hydrochloride, sulfate, phosphate, diphosphate, and
hydrobromate, and organic acid salts such as malate, maleate,
fumarate, tartarate, succinate, citrate, acetate, lactate,
methanesulfonate, p-toluenesulfonate, pamoate, salicylate and
stearate. Furthermore, the salt may be a salt with a metal such as
sodium, potassium, calcium, magnesium and aluminum or a salt with
an amino acid such as lysine. Furthermore, solvates such as
hydrates of the aforementioned compound or pharmaceutically
acceptable salts thereof also fall within the scope of the present
invention.
Dosage form of the drug of the present invention is not
particularly limited and can be suitably selected depending on the
therapeutic purpose. Specific examples thereof include tablet,
pill, powder, solution, suspension, emulsion, granules, capsule,
syrup, suppository, injection, ointment, patch, eye drop, nasal
drop and so forth. For the preparation, additives generally used in
usual hyperglycemia improving drugs as pharmaceutical carriers such
as excipients, binders, disintegrating agents, lubricants,
stabilizers, flavoring agents, diluents, surfactants and solvents
for injection can be used. Furthermore, so long as the effect of
the present invention is not degraded, the compound or composition
of the present invention can be used in combination with other
drugs having hyperglycemia improving effect.
Although the amount of the compound or the composition of the
present invention contained in the drug of the present invention is
not particularly limited and can be suitably selected, the amount
may be, for example, 0.001 to 10% by mass, preferably 0.01 to 1% by
mass, particularly preferably 0.05 to 1% by mass, in terms of the
amount of the compound of the present invention.
The drug of the present invention is useful for a therapeutic or
prophylactic treatment of a disease resulted from hyperglycemic
conditions such as, for example, diabetes and its associated
symptoms and conditions (likelihood of developing diabetes or
related conditions). In particular, it can also be used to prevent
onset of diabetes mellitus from hyperglycemic conditions.
The drug of the present invention contains the aforementioned agent
of the present invention (hyperglycemia improving agent) and is
useful for a therapeutic or prophylactic treatment of a disease
resulted from hyperglycemic conditions such as diabetes and its
associated symptoms and conditions (likelihood of developing
diabetes or related conditions). In particular, it can also be used
to prevent onset of diabetes mellitus from hyperglycemic
conditions. Furthermore, the drug of the present invention can cure
or prevent various diseases, complications and so forth resulted
from hyperglycemic conditions, and reduce risks of these diseases,
complications and so forth.
Examples of such various diseases and complications resulted from
hyperglycemic conditions include diabetic retinopathy, diabetic
nephropathy, diabetic neuropathy, diabetic gangrene, cerebral
apoplexy resulted from diabetes mellitus, myocardial infarction
resulted from diabetes mellitus and so forth.
The term "hyperglycemic conditions" refers to conditions that the
blood glucose levels are out of the normal ranges, and the normal
ranges are generally defined as a fasting blood glucose level of
110 mg/dl or lower, a blood glucose level 1 hour after 75 g glucose
load of 160 mg/dl or lower, and a blood glucose level 2 hours after
the same glucose load of 120 mg/dl or lower (Nihon Rinsho, No. 806,
Vol. 1, pp. 28-35, 2002). Furthermore, the drug of the present
invention is preferably used for a treatment of a patient with a
hemoglobin Alc level higher than normal, for example, a hemoglobin
Alc level of 5.8% or higher.
The administration time of the agent or drug of the present
invention is not particularly limited and can be suitably selected
according to the method for treating an objective disease.
Furthermore, the administration route is preferably determined
depending on the dosage form, age, sex and other conditions of
patients, severity of symptoms of patients and so forth.
The dose of the active ingredient in the agent of the present
invention is suitably selected depending on the dosing regimen,
age, sex, severity of disease, other conditions of patients and so
forth. The amount of the compound of the present invention as an
active ingredient is usually selected from the range of, preferably
0.01 to 10 mg/kg/day, more preferably 0.1 to 1 mg/kg/day, as a
tentative dose. Furthermore, when the composition of the present
invention is used, the dry weight of the composition is selected
from the range of, preferably 0.1 to 1000 mg/kg/day, more
preferably 1 to 100 mg/kg/day, as a tentative amount. In any case,
the dose can be ingested once daily or several times as divided
portions.
The agent or drug of the present invention or the active ingredient
thereof, i.e., the compound or the composition of the present
invention, can be added to food or drink. The form and property of
the food or drink are not particularly limited so long as the
effect of the active ingredient is not degraded, and the food or
drink can be orally ingested, and it can be produced in a
conventional manner by using raw materials usually used for food or
drink except that the aforementioned active ingredient is
added.
The amount of the compound or the composition of the present
invention contained in the food or drink of the present invention
is not particularly limited and can be suitably selected. For
example, the compound or the composition of the present invention
is contained in food or drink in an amount of 0.0001 to 1% by mass,
preferably 0.001 to 1% by mass, particularly preferably 0.005 to 1%
by mass, in terms of the amount of the compound of the present
invention.
The food or drink of the present invention can be used for various
applications utilizing the hyperglycemia improving effect. For
example, it can be used as food or drink suitable for those who are
getting concerned about their blood glucose levels, food or drink
useful for decreasing or eliminating risk factors of
lifestyle-related diseases such as diabetes mellitus.
As for the food or drink of the present invention, the expression
"improvement of hyperglycemia" means that improvement or prevention
of various health damages resulted from hyperglycemia, and
"prevention of hyperglycemia," "suppression of increase in blood
glucose level," "improvement of increase in blood glucose level,"
"prevention of increase in blood glucose level," "improvement of
high hemoglobin Alc level" and so forth are exemplified in the
present invention as terms having a meaning similar to that of the
aforementioned "improvement of hyperglycemia".
Furthermore, the food or drink of the present invention is useful
for a prophylactic treatment of a disease resulted from
hyperglycemic conditions such as diabetes mellitus and its
associated symptoms and conditions (likelihood of developing
diabetes or related conditions). In particular, it can also be used
to prevent onset of diabetes mellitus from hyperglycemic
conditions. Furthermore, the food or drink of the present invention
can be used for a prophylactic treatment of various diseases,
complications and so forth resulted from hyperglycemic conditions
and can decrease risks of these diseases, complications and so
forth.
Examples of such various diseases and complications resulted from
hyperglycemic conditions include diabetic retinopathy, diabetic
nephropathy, diabetic neuropathy, diabetic gangrane, cerebral
apoplexy resulted from diabetes mellitus, myocardial infarction
resulted from diabetes mellitus and so forth.
The food or drink of the present invention is preferably marketed
as food or drink attached with an indication that the food or drink
is used for improving hyperglycemia, for example, "food or drink
containing a compound having hyperglycemia improving effect
indicated as `For improving hyperglycemia,`" "food or drink
containing a plant extract indicated as `For improving
hyperglycemia,`" "food or drink containing Aloe vera extract
indicated as `For improving hyperglycemia`" and so forth.
Because the compound, the composition and others of the present
invention have a hyperglycemia improving effect, it is considered
that the indication of "improvement of hyperglycemia" also means
"suppression of increase in blood glucose level." Therefore, the
food or drink of the present invention can be indicated as "For
suppressing increase in blood glucose level." That is, the
aforementioned indication of "For improvement of hyperglycemia" may
be an indication of "For suppression of increase in blood glucose
level."
The wording used for such an indication as mentioned above is not
necessarily be limited to the expression "For improvement of
hyperglycemia" or "For suppression of increase in blood glucose
level", and any other wording expressing the effect of improving
hyperglycemia or suppressing increase in blood glucose level of
course falls within the scope of the present invention. As such a
wording, for example, an indication based on various uses allowing
consumers to recognize the effect of improving hyperglycemia or
suppressing increase in blood glucose level is also possible.
Examples include, for example, indications of "Suitable for those
who are getting concerned with blood glucose levels", "Useful for
decrease or elimination of risks of lifestyle-related diseases such
as diabetes mellitus".
The aforementioned term "indication" include all actions for
informing consumers the aforementioned use, and any indications
reminding or analogizing the aforementioned use fall within the
scope of the "indication" of the present invention regardless of
purpose, content, objective article, medium etc. of the indication.
However, the indication is preferably made with an expression that
allows consumers to directly recognize the aforementioned use.
Specific examples include actions of indicating the aforementioned
use on goods or packages of goods relating to the food or drink of
the present invention, actions of assigning, delivering, displaying
for the purpose of assigning or delivering or importing such goods
or packages of goods indicated with the aforementioned use,
displaying or distributing advertisements, price lists or business
papers relating the goods, or providing information including those
as contents with indicating the aforementioned use by an
electromagnetic method (Internet etc.) and so forth.
The indication is preferably an indication approved by the
administration etc. (for example, an indication in a form based on
an approval, which is qualified on the basis of any of various
legal systems provided by the administration), and it is
particularly preferably an indication on advertisement materials at
the sales spots such as packages, containers, catalogs, pamphlets
and POPs, others documents and so forth.
Examples of the indication further include, for example,
indications as health food, functional food, enteric nutritive
food, food for special dietary uses, food with nutrient function
claims, quasi-drug and so forth as well as indications approved by
the Ministry of Health, Labor and Welfare, for example, indications
approved on the basis of the system of food for specified health
uses and similar systems. Examples of the latter include
indications as food for specified health uses, indications as food
for specified health uses with qualified health claims, indications
of influence on body structures and functions, indications of
reduction of disease risk claims and so forth, and more precisely,
typical examples include indications as food for specified health
uses (especially indications of use for health) provided in the
enforcement regulations of Health Promotion Law (Japan Ministry of
Health, Labor and Welfare, Ministerial ordinance No. 86, Apr. 30,
2003) and similar indications.
EXAMPLES
The present invention will be explained more specifically with
reference to the following examples. However, the scope of the
present invention is not limited to these examples.
Preparation Example 1
Examples of preparation of
3-O-.beta.-D-glucopyranosyl-4-methylergost-7-en-3-ol from Aloe vera
will be described below.
3-O-.beta.-D-Glucopyranosyl-4-methylergost-7-en-3-ol was extracted
from Aloe vera and purified as described below.
In an amount of 100 kg of mesophyll (clear gel portion) of Aloe
vera was liquefied by using a homogenizer, added with 100 L of an
ethyl acetate/butanol mixture (3:1) and stirred.
The mixture was left standing overnight to separate the ethyl
acetate/butanol mixture and the aqueous layer, and the ethyl
acetate/butanol mixture was recovered. The extract from this ethyl
acetate/butanol mixture obtained by concentrating the ethyl
acetate/butanol mixture under reduced pressure weighed 13.5 g.
Hyperglycemia improving effect was evaluated for the aforementioned
aqueous layer and the extract from the ethyl acetate/butanol
mixture in diabetes model mice described later, and the effect was
observed for the extract from the ethyl acetate/butanol mixture.
Therefore, it was attempted to isolate and purify components in the
extract. First, the aforementioned extract was examined by thin
layer chromatography (Merck Ltd., Silica gel 60F254 and
RP-18F2543). As a result, an isolation method based on normal phase
silica gel column chromatography using a chloroform/methanol
mixture appeared to be suitable. Accordingly, a solution of 13 g of
the aforementioned extract dissolved in 1 mL of a
chloroform/methanol mixture (1:1) was loaded on a column filled
with 400 g of silica gel 60 (Merck Ltd.) to attain adsorption of
the components to the column, then the components were eluted with
a chloroform/methanol mixture by the stepwise gradient method, in
which the methanol concentration was increased stepwise (mixing
ratios of chloroform:methanol=100:1, 25:1, 10:1, 5:1 and 1:1), and
the eluate was fractionated for each mixing ratio of the
aforementioned mixture. The yields of crude purification products
obtained from the fractions after removing the solvent were 1.44,
3.0, 1.17, 1.28 and 2.27 g, respectively. It was confirmed by a
method using the aforementioned model animals that, among these
fractions, an active component existed in the fraction eluted with
the mixture of chloroform:methanol=5:1 (crude purification product
A). The existence of barbaloin or aloe-emodin was not confirmed by
thin layer chromatography analysis.
Furthermore, to isolate and purify the active component from the
aforementioned crude purification product A, this crude
purification product A was examined by using thin layer
chromatography (Merck Ltd., Silica gel 60F254 and RP-18F2543). As a
result, an isolation method based on reverse phase silica gel
column chromatography using methanol appeared to be suitable.
Accordingly, the aforementioned crude purification product A was
dissolved in 1 mL of a chloroform/methanol mixture (1:1) and loaded
on a column filled with 180 g of COSMOSIL 140 (Nacalai Tesque,
Inc.) to attain adsorption of the component to the column. Then,
elution was performed by successively using 600 mL of 85% methanol
solution, 600 mL of 95% methanol solution and 100 mL of 100%
methanol. 3-O-.beta.-D-Glucopyranosyl-4-methylergost-7-en-3-ol was
concentrated and isolated in a fraction eluted with 95% methanol
and weighed 370 mg after removing the solvent. Hereafter, this
product is referred to as compound 1.
Because the compound 1 showed an Rf value very close to that of
.beta.-sitosterol glucoside in an examination based on thin layer
chromatography, it was anticipated to be a glycoside in which 1
molecule of sugar bound to the aglycon moiety. Furthermore, to
examine the sugar composition of the compound 1, the compound 1 was
subjected to methanolysis, then made into a TMS derivative and
subjected to GC-MS measurement. As a result, in the measurement of
the TMS derivative for the sugar portion of the compound 1, it
showed main peaks at retention times of 14.28, 14.61 and 16.34
minutes, which were substantially consistent with the retention
times of the main peaks of the sample glucose (Nacalai Tesque,
Inc.), 14.27, 14.60 and 16.33 minutes. Furthermore, peaks
corresponding to the main peaks of the sample galactose (Kishida
Chemical Co., Ltd.) and the sample xylose (Kishida Chemical Co.,
Ltd.) were not observed. Thus, it was confirmed that the type of
the sugar contained in the compound 1 was glucose.
From the above results, it was estimated that the compound 1 was a
glycoside in which 1 molecule of glucose bound to the aglycon
moiety. However, when the compound 1 was measured by .sup.13C-NMR
(125 MHz, CDCl.sub.3), the existence of contaminants was confirmed.
Therefore, it was considered that further purification should be
required to determine its structure. Accordingly, the compound 1
was methanolyzed and then acetylated, and then the structure of the
aglycon moiety as well as the binding site of the aglycon moiety
and the sugar were confirmed. The method therefor will be described
below.
In an amount of 50 mg of the compound 1 was dissolved in methanol
(50 mL) containing 5% hydrochloric acid, refluxed with heating for
6 hours for methanolysis and dried to obtain a residue (about 30
mg). This residue was purified by silica gel column chromatography
(hexane:chloroform=9:1) to obtain a compound 2 (10 mg). This
compound 2 (5 mg) was added with acetic anhydride and pyridine (2
drops each) and heated at 70.degree. C. for 30 minutes for
acetylation, and then the solvent of the reaction mixture was
evaporated to obtain a compound 3. The results of the analysis of
this compound 3 by GC-MS and .sup.13C-NMR (125 MHz, CDCl.sub.3) are
shown in FIGS. 1 and 2, respectively. The measurement conditions
and results are as follows. 3-Acetoxy-4-methylergost-7-ene used as
a reference substance was prepared by extracting aloe, purifying
the extract, confirming the structure of the purified product by
.sup.13C-NMR and acetylating the same.
[.sup.13C-NMR spectrum (d values, in CDCl.sub.3)]; C-1:36.8,
C-2:27.3, C-3:78.7, C-4:37.0, C-5:46.9, C-6:26.8, C-7:117.4,
C-8:139.4, C-9:49.7, C-10:34.9, C-11:21.6, C-12:39.7, C-13:43.6,
C-14:55.1, C-15:23.1, C-16:28.2, C-17:56.3, C-18:12.0, C-19:14.2,
C-20:36.5, C-21:19.0, C-22:33.9, C-23:30.6, C-24:39.1, C-25:32.6,
C-26:20.4, C-27:18.4, C-28:15.6, C-29:15.3
[GC-MS]
Apparatus: GC-17A/GCMS5050A (SHIMADZU) GC column: NEUTRA BOND-5 (GL
Scienses) Column temperature: 100.degree. C. (2
min).fwdarw.(10.degree. C./min).fwdarw.300.degree. C. (28 min)
Injection temperature: 250.degree. C. Carrier gas: He (1.3 mL/min)
Interface temperature: 300.degree. C. MS mode: EI Ionization
energy: 70 eV [Results] Reference substance:
3-acetoxy-4-methylergost-7-ene: tR [min]=39.4; m/z 456 [M].sup.+,
441 [M-CH.sub.3].sup.+, 396 [M-AcOH].sup.+, 381
[M-CH.sub.3-AcOH].sup.+ Compound 3: tR [min]=39.2; m/z 456
[M].sup.+, 441 [M-CH.sub.3].sup.+, 396 [M-AcOH].sup.+, 381
[M-CH.sub.3-AcOH].sup.+
The results of the NMR measurement of the compound 3 were
consistent with the values of 3-acetoxy-4-methylergost-7-ene shown
in a literature (Yukagaku (Oil Chemistry), Vol. 36, No. 5, pp.
301-319, 1987). These results revealed that the compound 2 was
4-methylergost-7-en-3-ol. Furthermore, as a result of FAB-MS
measurement, the molecular weight of the compound 1 was found to be
576. When the compound 2 (aglycon moiety) and glucose were
condensed, the molecular weight of the obtained compound was 414
(compound 2)+180 (glucose)-18 (water)=576, which was consistent
with the molecular weight of the compound 1.
The above results revealed that the compound 1 had a structure of 3
O-.beta.-D-glucopyranosyl-4-methylergost-7-en-3-ol.
The molecular formulas, molecular weights and chemical formulas of
the compounds are shown below.
(Compound 1)
Molecular formula: C.sub.35H.sub.60O.sub.6 Molecular weight: 576
Chemical formula: The following chemical formula (1)
##STR00002## (Compound 2) Molecular formula: C.sub.29H.sub.50O
Molecular weight: 414 Chemical formula: The following chemical
formula (2)
##STR00003## (Compound 3) Molecular formula:
C.sub.31H.sub.52O.sub.2 Molecular weight: 456 Chemical formula: The
following chemical formula (3)
##STR00004##
Preparation Example 2
Mesophyll (clear gel portion) of Aloe vera was dried by heating,
0.3 g of disrupted dry Aloe vera powder was added with 60 mL of 60,
80 or 100% ethanol and refluxed by heating at 60.degree. C. for 1
hour. The extract was centrifuged at 1500 rpm for 20 minutes, and
the supernatant was concentrated under reduced pressure to
completely remove ethanol and thereby obtain a crude extract. The
dry weights of the crude extracts obtained by extraction using 60,
80 and 100% ethanol were 65, 42 and 18 mg, respectively. It was
confirmed by thin layer chromatography that these crude extracts
contained 3-O-.beta.-D-glucopyranosyl-4-methylergost-7-en-3-ol.
Preparation Example 3
Mesophyll (clear gel portion) of Aloe vera was dried by heating,
0.3 g of dry crushed Aloe vera powder was added with 60 mL of water
and refluxed by heating at 95.degree. C. for 5 hours. The extract
was centrifuged at 1500 rpm for 20 minutes, and the supernatant was
lyophilized to obtain 75 mg of a crude extract. It was confirmed by
thin layer chromatography that this crude extract contained
3-O-.beta.-D-glucopyranosyl-4-methylergost-7-en-3-ol.
Preparation Example 4
Mesophyll (clear gel portion) of Aloe vera was dried by heating,
disrupted and dried, 21 kg of Aloe vera powder thus prepared was
added with 90 L of a chloroform/methanol mixture (2:1), then
immersed overnight in the mixture at room temperature and collected
by filtration, and the residue obtained by the filtration was added
with 90 L of chloroform/methanol mixture (2:1) again. This
procedure was repeated 4 times in total. The obtained filtrate (350
L) was concentrated at 28.degree. C. to finally obtain 784 g of a
crude extract. In an amount of 780 g of this crude extract was
added with 2 L of a chloroform/methanol mixture (2:1), stirred for
1 hour and filtered to recover the chloroform/methanol mixture
layer (A). The residue obtained by the filtration was successively
added with 2.5 L of water and 2 L of ethyl acetate and stirred for
1 hour, and the ethyl acetate layer (B) was recovered. The
remaining aqueous layer was added with 5 L of chloroform again and
stirred for 1 hour, and the chloroform layer (C) was recovered.
The recovered organic solvent extracts A, B and C were mixed,
concentrated at 23.degree. C. and loaded on a silica gel column
[glass column, 52 mm.times.350 mm, packed material: IR-63/210-W
(Daiso Co., Ltd.)]. Subsequently, while monitoring the eluate by
thin layer chromatography, 10 L of a hexane/chloroform mixture
(1:1), 10 L of chloroform, 20 L of a chloroform/methanol mixture
(10:1) and 20 L of a chloroform/methanol mixture (5:1) were passed
through the column in this order, and a fraction 1 (about 1 L),
fraction 2 (about 1.5 L), fraction 3 (about 1.5 L) and fraction 4
(about 1.5 L) were recovered in the order of the used elution
solvents.
It was confirmed by thin layer chromatography that, among these,
the fraction 3 contained the objective glycoside, and then the
solvent of the fraction 3 was removed to obtain 131.6 g of a crude
extract. In an amount of 130 g of this crude extract was loaded on
a silica gel column [glass column, 70 mm.times.500 mm, packed
material: SP-60-40/60 (Daiso Co., Ltd.)] again and eluted
successively with 10 L of a chloroform/methanol mixture (30:1), 50
L of a chloroform/methanol mixture (20:1), 10 L of a
chloroform/methanol mixture (10:1) and 10 L of a
chloroform/methanol mixture (1:1) as elution solvents under
conditions of a pressure of 10 kgfcm.sup.-2 and a flow rate of 40
mL/min. The eluates were fractionated as 100-mL fractions by using
a fraction collector to collect fractions 1 to 8.
The collected fractions were examined by thin layer chromatography,
and as a result, it was revealed that the objective glycoside and
contaminants existed in the fraction 7. Therefore, this fraction
was concentrated, loaded on a silica gel column [glass column, 70
mm.times.500 mm, packed material: SP-60-40/60 (Daiso Co., Ltd.)]
again, and successively eluted with 10 L of a chloroform/methanol
mixture (20:1) and 10 L of a chloroform/methanol mixture (10:1) as
elution solvents under conditions of a pressure of 10 kgfcm.sup.-2
and a flow rate of 40 mL/min. As a result, 25.3 g of
3-O-.beta.-D-glucopyranosyl-4-methylergost-7-en-3-ol was prepared,
which was the objective glycoside contained in the elution fraction
of the chloroform/methanol mixture (10:1).
Test Example 1
This test was performed in order to evaluate the hyperglycemic
condition improving effect of
3-O-.beta.-D-glucopyranosyl-4-methylergost-7-en-3-ol.
(1) Preparation of Sample
The 3-O-.beta.-D-glucopyranosyl-4-methylergost-7-en-3-ol produced
in Preparation Example 1 mentioned above was used as a test
sample.
(2) Test Method
As type-II diabetes model mice, 6-week old male db/db mice
(purchased from Clea Japan, Inc.) were used. These mice were
divided into groups, each consisting of 7 animals. The test sample
was dissolved in DMSO, and the concentration of
3-O-.beta.-D-glucopyranosyl-4-methylergost-7-en-3-ol was adjusted
to 15 .mu.g/mL with physiological saline. The final DMSO
concentration was adjusted to 0.2%. The type-II diabetes model mice
were orally administered 1 mL of the test sample solution once a
day everyday with a sonde. A solution that did not contain the test
sample was used as a negative sample. Fasting blood glucose levels
and random blood glucose levels were measured over time using
Antsense II (Bayer-Sankyo Co., Ltd.). The fasting blood glucose
levels were measured after 15 hours of fasting.
(3) Hyperglycemia Improving Effect
Changes over time in random blood glucose levels and fasting blood
glucose levels during the test sample administration period are
shown in FIGS. 3 and 4. In the mice administered with the negative
sample, rapid increase were observed in both the random blood
glucose levels and fasting blood glucose levels, whereas effect of
suppressing the increases in blood glucose levels was clearly
observed in the mice repeatedly administered with the test
sample.
Test Example 2
This test was performed in order to evaluate the hemoglobin Alc
lowering action of
3-O-.beta.-D-glucopyranosyl-4-methylergost-7-en-3-ol.
(1) Preparation of Sample
The 3-O-.beta.-D-glucopyranosyl-4-methylergost-7-en-3-ol produced
in Preparation Example 1 mentioned above was used as a test
sample.
(2) Test Method
As type-II diabetes model mice, 6-week old male db/db mice
(purchased from Clea Japan, Inc.) were used. These mice were
divided into groups, each consisting of 7 animals. The test sample
was dissolved in DMSO, and the concentration of
3-O-.beta.-D-glucopyranosyl-4-methylergost-7-en-3-ol was adjusted
to 1, 5 and 15 .mu.g/mL with physiological saline. The final DMSO
concentration was adjusted to 0.2%. The type-II diabetes model mice
were orally administered with 1 mL of the test sample solution once
day everyday with a sonde. A solution that did not contain the test
sample was used as a negative sample. On the 35th day from the
start of the administration, hemoglobin Alc levels were measured by
using DCA 2000 (Bayer-Sankyo Co., Ltd.).
(3) Hemoglobin Alc Lowering Action
The measurement results of hemoglobin Alc levels on the 35th day
from the start of the administration are shown in Table 1. In
comparison with the hemoglobin Alc levels after administration of
the negative sample, statistically significant decreases in the
hemoglobin Alc levels were observed after the repetitive
administration of 5 or 15 .mu.g of the test sample, indicating
existence of blood glucose level control effect over a long period
of time. Further, during the administration period, there was no
case showing adverse side effect symptoms or acute hypoglycemic
conditions after the administration, and no abnormality was
observed in body weight and pathological findings.
TABLE-US-00001 TABLE 1 Hemoglobin Alc relative levels (%) 35th day
from the Samples administration p value Test sample (1 .mu.g) 98.6
.+-. 7.3 Test sample (5 .mu.g) 89.6 .+-. 7.9* 0.017 Test sample (15
.mu.g) 73.5 .+-. 8.6* 0.00001 negative sample 100 *statistical
significance was observed.
Test Example 3
This test was performed in order to examine the hemoglobin Alc
lowering action and the dose of the extracted crude purification
product A containing
3-O-.beta.-D-glucopyranosyl-4-methylergost-7-en-3-ol derived from
Aloe vera.
(1) Preparation of Sample
The extracted crude purification product A containing
3-O-.beta.-D-glucopyranosyl-4-methylergost-7-en-3-ol produced in
Preparation Example 1 mentioned above was used.
(2) Test Method
As type-II diabetes model mice, 6-week old male db/db mice
(purchased from Clea Japan, Inc.) were used. These mice were
divided into groups, each consisting of 7 animals. The test sample
was dissolved in DMSO, and the concentration of the extracted crude
purification product A was adjusted to 25, 100 and 200 .mu.g/mL
with physiological saline. The final DMSO concentration was
adjusted to 0.2%. The type-II diabetes model mice were orally
administered with 1 mL of the test sample solution once a day
everyday with a sonde. A solution that did not contain the test
sample was used as a negative sample. On the 35th day from the
start of the administration, hemoglobin Alc levels were measured by
using DCA 2000 (Bayer-Sankyo Co., Ltd.).
(3) Blood Glucose Level and Hemoglobin Alc Level
The measurement results of hemoglobin Alc levels on the 35th day
from the start of the administration are shown in Table 2. In
comparison with the hemoglobin Alc level of the negative test,
decreases in the hemoglobin Alc level were observed after
repetitive administration of 100 or 200 .mu.g of the test sample,
and thus existence of blood glucose level control effect over a
long period of time was statistically significantly demonstrated.
Further, during the administration period, there was no case
showing adverse side effect symptoms or acute hypoglycemic
conditions after administration, and no abnormality was observed in
body weight and pathological findings.
TABLE-US-00002 TABLE 2 Hemoglobin Alc relative levels (%) 35th day
from Samples the administration P value Test sample (crude 92.5
.+-. 7.1 0.1571 purification product 25 .mu.g) Test sample (crude
84.9 .+-. 8.2* 0.0275 purification product 100 .mu.g) Test sample
(crude 82.0 .+-. 8.6* 0.0129 purification product 200 .mu.g)
negative sample 100 *statistical significance was observed.
The compound of the present invention can be safely administered or
ingested without causing acute hypoglycemia and has a long-term
blood glucose level control action lowering the hemoglobin Alc
level. Further, the composition of the present invention can be
produced by using a plant of the family Liliaceae, a plant that can
be safely ingested from experiential viewpoint for food and is
readily available, for example, a plant of the genus Aloe or
Allium. Further, the composition of the present invention contains
an effective amount of the compound of the present invention, and
in a preferred embodiment thereof, it does not contain barbaloin or
aloe-emodin, which are unfavorable components as a drug, food or
drink.
* * * * *